Hydrophilic macroporous three dimensional copolymers of hydroxyalkyl acrylates or methacrylates with crosslinking agents and the method of their manufacturing

ABSTRACT

The invention pertains to hydrophilic macroporous threedimensional copolymers of hydroxyalkyl methacrylates or hydroxyalkyl acrylates (C 1  -C 4  hydroxyalkyl) with crosslinking monomers which contain covalently attached saccharides via an O-glycosidic bond. The invention further pertains to the method of manufacturing of these copolymers by the direct reaction of the basic three dimensional copolymer containing hydroxyl groups with saccharides or their derivatives in an inert organic solvent under catalysis of HCl, BF 3 , or their mixtures at 20°-100° C. Saccharides and their derivatives are selected from the group comprising monosaccharides, oligosaccharides, deoxy sugars, amino saccharides, acylated saccharides, ether or halogen derivatives of monosaccharides and oligosaccharides, and the like. The invention also pertains to the application of these macroporous three dimensional copolymers with saccharides as sorbents and materials for the affinity chromatography for isolation of physiologically effective materials which contain free bonding sites for saccharides and are able to form specific complexes with saccharides.

The invention pertains to hydrophilic macroporous three dimensionalcopolymers of hydroxyalkyl methacrylates or hydroxyalkyl acrylates withcrosslinking comonomers containing covalently bound saccharides and tothe method of their manufacturing by the direct reaction of the polymerswith saccharides under catalysis of Lewis acids.

Hydrophilic macroporous copolymers of hydroxyalkyl methacrylates andhydroxyalkyl acrylates with alkylene dimethacrylates or alkylenediacrylates prepared by a suspension copolymerization of monomers in thepresence of inert organic compounds in an aqueous or organic dispersionmedium are known from the Czechoslovak Pat. No. 150,819, whichcorresponds to the British Pat. No. 1,370,477, and from the CzechoslovakPat. No. 148,828, which corresponds to the British Pat. No. 1,331,087.Their outstanding mechanical properties, spherical shape of particles,and a macroporous structure, allowing to penetrate even considerablylarge molecules into the pores of carrier, led to the extensiveapplications of these materials in the gel, ion-exchange, adsorption,and affinity chromatography using water or aqueous solutions as aneluent. Utilizing of hydroxyalkyl methacrylate and hydroxyalkyl acrylatecopolymers seems to be very perspective even in the hydrophobicchromatography which employs interactions between the lipophilic part ofmolecules which are separated and the relatively nonpolar matrix ofcarrier, the hydrophility of which is caused by the presence ofnonionogenic hydroxyl groups in the side chains of copolymers.

However, this possible hydrophobic interaction between the carrier andmolecules dissolved in water became undesirable for some applications.This holds everywhere the unambiguous interaction between the functionalgroups of carrier and an interacting compound is required (ion-exchangeand affinity chromatography) or where the sorption interaction should besuppressed totally, as it is in the case of gel chromatography. Theinner surface of macroporous copolymer has to be then hydrophilized by achemical transformation of hydroxyl groups. The strongesthydrophilization may be attained by substitution of hydroxyl withionogenic functional groups, e.g. by the methods according to theCzechoslovak Pat. Nos. 171,962; 171,963 (U.S. Pat. No. 3,991,018)177,507 or the British Pat. Nos. 3,500,532 and 1,499,134. However, alsothe presence of ionogenic functional groups is undesirable in numerouscases.

In some carriers designated for further chemical transformations, a lowcontent of hydroxyl groups capable of transformation is disadvantageousbecause it causes a lower capacity of the final product than it wasrequired. This comes true above all if the high mechanical stability ofcarrier is necessary (in microparticular sorbents for application inhigh-performance liquid chromatography) and, consequently, where thecopolymers have to be prepared with a high content of a crosslinkingagent, which does not contain hydroxyl groups. In these cases, thesubstitution of hydroxyl groups by the molecule of saccharide representsthe solution of the required higher capacity for polymeranalogoustransformations. In addition to this, the chemically bound sacchariderepresents, both in the basic form and the derivatized form, a basis fornumerous polymeranalogous reactions where the glycosylated polymer isthe reagent. Another advantage of the carrier containing chemicallybound saccharides may be seen in the regenerability of carrier, becausethe original carrier which may be again modified is recovered by ahydrolytic cleavage under rather drastic conditions.

The most important carriers, among those which have been appliedrecently, are modified natural polysaccharides, glycoproteins linked tovarious carriers, and also sugars or their derivatives bound by acovalent bond to various matrices.

In the first group, the commercially available polysaccharides in acrosslinked form proved especially useful. Among them there are reckonedabove all polydextrans crosslinked with epichlorohydrine, agarose gels,crosslinked gum arabic, chitin, etc. Glycoproteins occur mostly in asoluble form and require bonding to a natural or syntetic carrier forthe purpose of affinity chromatography. The third group of sorbentsincludes monosaccharides or oligosaccharides bound most often to apolysaccharide carrier either directly or through a distance coupling(spacer) in the form of the reactive amino derivative. Another methodemploys the reactive functional groups of carrier or of the reactiveinterlink-divinylsulfone. Application of acrylamide carriers representsan important progress. They are prepared in three different ways: bymodification of commercial preparations, by the synthesis of gels withactive ester groups incorporated by polymerization and able to reactwith amino sugars, or by copolymerization of omega-unsaturated aliphaticglycosides with acrylamide.

The large number of various types of carriers gives evidence of thefact, that only few of them have nearly ideal properties. Modifiednatural polysaccharides are most easily available, however their choiceis limited and they possess a low resistance against the attack ofmicroarganisms. A disadvante consists also in the fact, that some ofthese carriers cannot be prepared with a sufficient porosity whilemaintaining the satisfactory mechanical stability. The broaderapplication of glycoproteins bound to a carrier is limited also by thecomplexity and high cost of preparation of affinity-chromatographymaterials as well as their low resistance to decomposition.

Many of the above disadvantages do not occur with acrylamide gels, whichare substantially more resistant to the action of chemicals andmicroorganisms and allow the repeated use.

However, all above said materials are prepared in the form of soft,homogeneously crosslinked particles considerably swelling in aqueoussolvents and changing their volume with the change of pH or ionicstrength. They do not allow application of higher pressures and higherthrough-flow rates. From the standpoint of diffusion control of thekinetics of formation of affinity chromatography complexes and of theirdissociation, these materials are not too much suitable.

An objective of this invention are hydrophilic macroporous threedimensional copolymers of hydroxyalkyl acrylates or hydroxyalkylmethacrylates, which contain 1-4 carbon atoms in the hydroxyalkyl group,with crosslinking agents selected from the group comprising divinyl orpolyvinyl monomers, which copolymers are modified on the surface ofsaccharides or their derivatives linked by the covalent O-glycosidicbond and selected from the group comprising monosaccharides andoligosaccharides, deoxy sugars, amine sugars, acylated saccharides, andether or halogen derivatives of monosaccharides and oligosaccharides.

Alkylene diacrylates, alkylene dimethacrylates, polyglycol diacrylates,polyglycol dimethacrylates, alkylene-bis-acrylamides, divinylbenzene,and others are advantageously used as crosslinking agents. Also othercrosslinking agents may be employed including the copolymerizablecompounds which contain two or more ethylenic double bonds ot two ormore nonconjugated vinyl groups of formula CH₂ ═CH--, as divinyltoluene,trivinylbenzene, divinylnaphthalene, divinylxylene, divinylethylbenzene,divinyl ether, divinyl ketone, allyl acrylate, and the like.

The crosslinking agent is added in the amount of at least 15 wt.%,advantageously in the amount of 30-50 wt.% in respect to monomericmixture.

The polymerization of hydroxyalkyl acrylates orhydroxyalkylmethacrylates with crosslinking agents is carried out underthe conditions of suspension radical polymerization in an aqueous mediumin the presence of inert organic compounds, the character andconcentration of which influence the distribution of pores of theobtained products, and in the presence of suspension stabilizers.Cyclohexanol, benzyl alcohol, cyclohexylamine, dodecyl alcohol, n-octylalcohol, or their mixtures are advantageously used as the inert organiccompounds. As the suspension stabilizers may serve, for example,polyvinylpyrrolidone, poly(vinyl alcohol), partially hydrolyzedpoly(vinyl acetate), or some other natural polymeric materials (starch,pectins).

Spherical particles are formed in the suspension polymerization. Theparticles of the desirable distribution of size can be prepared bychoosing the conditions of the reaction (the size, shape, andrevolutions of the stirrer, concentrations of suspension stabilizer, andthe like.

The method of manufacturing of the materials according to the inventionrepresents the substitution of hydroxyl groups with saccharides underformation of the O-glycosidic bond, which is carried out in an inertorganic solvent at temperature 20°-100° C. under catalysis of hydrogenchloride, boron trifluoride, or their mixtures.

The reaction of saccharides with macroporous copolymers proceeds verysmoothly by mixing the suspension of a carrier in an organic inertsolvent (advantageously dioxan or tetrahydrofuran) which contains theappropriate amount of hydrogen chloride, boron trifluoride, or theirmixture. The solvents have to be dry and similarly is dried also HCl orBF₃ used for saturation. A finely ground saccharide is added into thesuspension and the mixture is allowed to react under stirring or shakingat the ambient or elevated temperature (20°-100° C.). The presence ofwater in the reaction components reduces the yield of bonding reaction.Because the formation and hydrolysis of O-glycosidic bond represent anequilibrium reaction, the optimum operation conditions for bonding themaximum amount of saccharides were investigated. The increasingtemperature up to 60°-70° C. increases the yield moderately. Furtherincrease of temperature up to 100° C. led to the decrease of yield. Themore expressive effect on the reaction yield has the concentration ofcatalyst (HCl or BF₃) which was followed up to 20 wt.%. The best resultswere achieved with the mixed catalyst HCl+BF₃ where hydrogen chlorideacts apparently by a cocatalytic effect. On the other hand, thecatalysis with H₃ PO₄, P₂ O₅, H₂ SO₄, and other acids proved ineffectiveunder the reaction conditions described.

Analysis of the content of bound saccharides was carried out by themethod according Dubois after hydrolysis.

The materials prepared according to this invention combine theoutstanding mechanical and hydrodynamic properties of the threedimensional hydroxyalkyl acrylate and methacrylate copolymers with agood hydrophility and suitable interaction properties comparable withpolydextrans, agarose and cellulose materials, which find now thebroadest application above all in biology and biochemistry.

The copolymers according to the invention have a high mechanicalstability and pressure resistance, are relatively resistant towards bothacid and alkaline hydrolysis, oxidation, and resistant to the effect oforganic solvents. The copolymers swell in aqueous solutions only verylittle and the particles do not change their size with the changingionic strength and pH.

The manufacturing method is not technologically demanding and providesreproducible yields in a broad range of reaction conditions.

The solvents, which are used for the reaction, have to dissolve hydrogenchloride and boron trifluoride well, without reacting with them atlaboratory or moderately elevated temperatures. It is further desirable,that the solvents have the polar character which enables at least apartial dissolution of saccharides chosen for bonding to hydroxyalkylacrylate or hydroxyalkyl methacrylate carriers. Dioxan ortetrahydrofuran are advantageously used.

The main advantage of the manufacturing method for glycosyl derivativesof hydroxyalkyl methacrylate and hydroxyalkyl acrylate copolymersconsists in its simplicity and rapidness which highly exceeds allmethods used until now for preparation of the similar type of sorbents.The mildness of preparation conditions for the materials according tothe invention, the easily accessible reaction components, the resistanceof matrix towards hydrolysis and microorganisms, simple handling andstorage are further merits.

The modified carriers according to the invention represent basis for anew series of chromatographic materials with a high variety of usesabove all for the separation of biopolymers and their fragments or forapplication in bonding of biologically active compounds designated forcatalysis.

The copolymers according to the invention may be used, for example, asspecific sorbents for isolation of physiologically effective compoundswhich contain free bonding sites and thus form with them specificcomplexes. Active compounds are selectively adsorbed from a mixture withinactive ballast. admixtures on the copolymers according to thisinvention and are released from the formed complexes by exchange withthe corresponding haptene or by elution with a suitable buffer solution.

The objective of the invention is further illustrated in examples, whichhowever do not limit the scope of invention by any means.

EXAMPLE 1

A copolymer (5 g) of hydroxyethyl methacrylate with 39 wt.% of ethylenedimethacrylate, prepared by the suspension polymerization in an aqueousmedium in the presence of inert solvent system consisting ofcyclohexanol and lauryl alcohol (9:1) and having the exclusion limit of300,000 daltons, was allowed to swell in a 500 ml reaction flask in 70ml of dioxan containing 17.5 wt.% of hydrogen chloride for 7 hours atlaboratory temperature. Vacuum was then shortly applied to the mixture.Anhydrous finely powdered D-glucose (3 g) was added to the suspensionwhich was then shaken for 14 hours at laboratory temperature. After thattime, the reaction was stopped by pouring the reaction mixture into 1liter of deionized water. The sugar derivative of gel was immediatelyseparated by a sintered-glass filter and washed with deionized water tothe neutral reaction and negative test on sugars. The glycosylated gelwas eventually washed with ethanol, acetone and ether and dried attemperature 50° C. in vacuo to the constant weight. The content of boundsugar amounted to 13.5 wt.%.

EXAMPLE 2

Similarly as in Example 1, 4.2 g of the hydroxyethyl methacrylatecopolymer was allowed to swell in 70 ml of dioxan saturated with gaseoushydrogen chloride to 5 wt.%. Then, 1.02 g of D-glucose was added and thesuspension was shaken at temperature 45° C. for 10 hours; the suspensionwas poured into 1 liter of deionized water and further worked out as inExample 1. The content of bound sugar was 6.42 wt.%.

EXAMPLE 3

The copolymer (3.5 g) of 2-hydroxyethyl methacrylate with ethylenediacrylate of the molecular weight exclusion limit 300,000 daltons wasallowed to swell for 12 hours in 60 ml of anhydrous dioxan whichcontained 6 wt.% of hydrogen chloride. N-Acetyl-D-glucosamine (1.3 g)was then added and the mixture was shaken at temperature 35° C. for 10hours. The suspension was poured into 1 liter of deionized water andfurther worked out in the same way as in Example 1. The content of boundamino sugar was 8.15 wt.%.

EXAMPLE 4

The copolymer of 2-hydroxyethyl acrylate with ethylene dimethacrylate (5g) of the exclusion limit 250,000 daltons was allowed to swell in 70 mlof dioxan, saturated with boron trifluoride to 7 wt.%, for 12 hours atlaboratory temperature. Anhydrous D-glucose (3 g) was added to thereaction suspension and the reaction mixture was shaken for 8.5 hours attemperature 50° C. The reaction was stopped by pouring the mixture into1 liter of deionized water and further worked out in the same way as inExample 1. The content of linked amino sugars was 12.4 wt.%.

EXAMPLE 5

The reaction was carried out analogously as in Example 1, with thedistinction that the reaction temperature was increased to 70° C. at thehydrogen chloride concentration in dioxan 6%. The final content ofcovalently bound D-glucose was 6.25 wt.%.

EXAMPLE 6

The copolymer of 2-hydroxyethyl methacrylate with ethylenedimethacrylate (5 g) of the exclusion limit 300,000 daltons was mixedwith 66 ml of dioxan and saturated with hydrgen chloride and borontrifluoride to the concentration 3.4 and 3.3 wt.%, respectively. Thesuspension was allowed to swell for 6.6 hours at the laboratorytemperature with occasional shaking. Then, 3 g of anhydrous and finelyground D-glucose was added and the mixture was shaken at 35° C. for 1hour. The reaction mixture was then kept for 12 hours at laboratorytemperature under continuous shaking. The reaction was stopped bypouring the reaction mixture into 1 liter of deionized water and thepolymer was further worked out in the same way as in Example 1. Thecontent of bound sugar was 17.6 wt.%.

EXAMPLE 7

Bonding of disaccharide was carried out similarly as in Example 1, withthe distinction that 3.26 g of finely ground maltose was used in thereaction and the reaction mixture was agitated on a laboratory shakerfor 10 hours at temperature 40° C. The gel with bound disaccharide wasworked out in the same way as in Example 1. The content of bound maltosewas 9.7 wt.%.

EXAMPLE 8

The experiment was carried out in the same way as in Example 6, with thedistinction that 6-O-methylglucose was used instead of D-glucose. Thefinal content of the bound ether derivative of glucose was 15.2 wt.%.

EXAMPLE 9

The bound halogen derivative of saccharide was prepared as in Example 6,with the distinction that 3 g of 6-fluoroglucose was used for thereaction. The resulting product was worked out in the same way as inExample 6 and the content of bound 6-fluoroglucose was 14.78 wt.%.

EXAMPLE 10

The lyophilized active fraction of proteins from soya beans (Glycinesoja) (400 mg) was dissolved in 4 ml of water. The undissolved residueswere removed by centrifugation and sodium chloride was added to thesupernatant up to concentration 0.9%. The solution of 4 ml of the activeprotein fraction (the determined activity of 1% solution against thetryptinised blood cells of group A₁ was 64) was applied to the column ofpoly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (mol. wt.exclusion limit 300,000 daltons) with 7.66 wt.% of bound D-galactose(weight of the dry carrier 4 g), which was equilibrated with physiologicsaline. After soaking of the sample, the column was eluted with salineat the flow rate 8 ml/h and 4 ml fractions were collected. The eluate infractions 3-5 was active in haemaglutination. From the fraction 20, theelution started with the 0.2 M solution of D-galactose in saline. Thefractions 22-24 were combined, dialysed for 48 hours against 4×2 literswater and lyophilised. The yield was 6.2 mg; activity of 1% solution4096.

EXAMPLE 11

The solution containing 200 mg of active proteins from lentils (Lensesculenta) (activity of 1% solution 64) in 2 ml of physiologic salinewas applied to the column (1×30 cm) containing poly(2-hydroxyethylmethacrylate-co-ethylene dimethacrylate) (mol. wt. exclusion limit300,000 daltons) with 8.25 wt.% of covalently bound D-glucose which wasequilibrated with saline. After soaking of the sample, the column waseluted with saline at the flow rate 8 ml/h and 4 ml fractions werecollected. The eluate in fractions 3-5 was active in haemaglutination.The elution with 0.2 M solution of D-glucose in saline was started fromthe 25th fraction. The fractions 28-30 were combined, dialysed for 48hours against 4×2 l of water, and lyophilised. The yield was 5.8 mg;activity of 1% solution 256.

EXAMPLE 12

The solution of 250 mg of the active fraction of proteins from lentils(Lens esculenta) (activity of 1% solution 64) in 2.5 ml of physiologicsaline was applied to the column (1×30 cm) containing 4 g ofpoly(2-hydroxyethyl acrylate-co-ethylene diacrylate) (mol. wt. exclusionlimit 500,000 daltons) with 9.0% of covalently bound mannose, which wasequilibrated with saline. After soaking of the sample, the column waseluted with saline at the flow rate 8 ml/h and 4 ml fractions werecollected. The eluate in fractions 3-5 was active in haemaglutination.The elution with 0.2 M solution of D-mannose in saline was started fromthe 28th fraction. The fractions 31-35 were combined, dialysed for 48hours against 4×3 l of water and lyophilised. The yield was about 1 mg.

EXAMPLE 13

The solution of 300 mg of the lyophilised extract from seeds of Ricinussommunis L. (activity of 1% solution 8192) in 3 ml of physiologic salinewas applied to the column (1×30 cm) packed with 4 g of sphericalmacroporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)(mol. wt. exclusion limit 1,000,000 daltons) with 7.66 wt.% ofchemically bound D-galactose, which was equilibrated with saline. Aftersoaking of the sample, the column was eluted with saline at the flowrate 8 ml/h and 4 ml fractions were collected. The eluate in the 3rdfraction was active in haemaglutination. The elution with 0.2 M solutionof D-galactose in saline was started from the 21st fraction. Thefractions 24-28 were combined, dialysed for 48 hours against 4×3 l ofwater, and lyophilised. The yield was 77.2 mg; activity of 1% solutionwas 32,768.

EXAMPLE 14

The solution containing 700 mg of the haemaglutination active fractionof proteins from seeds of Ulex europaeus L. in 6 ml of 0.15 phosphatebuffer solution of pH 7.9 was applied to the column (1×30 cm) containing4 g of spherical macroporous poly(2-hydroxyethylmethacrylate-co-ethylene dimethacrylate) (mol. wt. exclusion limit300,000 daltons) with 5.42 wt.% of bound L-fucose, which wasequilibrated with the same buffer solution. After soaking of the sample,the column was eluted with the same buffer solution at the flow rate 15ml/h and 5 ml fractions were collected. The elution with 50 ml of 0.2 Msolution of L-fucose in the above buffer solution was started from the40th fraction and, after soaking of the L-fucose solution, it wascontinued with the phosphate buffer solution at the unchanged flow rate.The fractions 43-48 were combined dialysed for 48 hours against 4×5 l ofwater, and lyophilised. The yield was 7.1 mg.

EXAMPLE 15

The haemaglutination active extract (5 ml) obtained from 1.5 g ofalbumine glands of snail (Helix pomatia) was applied to the column (1×20cm) containing 3.1 g of spherical macroporous poly(2-hydroxyethylmethacrylate-co-ethylene dimethacrylate) with 8.15 wt.% of covalentlybound N-acetyl-D-glucosamine, which was equilibrated with physiologicsaline. After soaking of the sample, the column was eluted with salineat the flow rate 8 ml/h and 4 ml fractions were collected. The eluate ofno fraction was haematoglutination active. The elution with 0.2 Msolution of N-acetyl-D-glucosamine in saline was started from the 23rdfraction and with the glycine buffer solution of pH 2.7 from the 38thfraction. The fractions 26-29 were collected, dialysed for 48 hoursagainst 4×3 l of water and lyophilized. The yield was 16.3 mg; activityof 1% solution 32,768.

EXAMPLE 16

The haemaglutination active extract (20 ml) from Jack beans meal(activity of 1% solution against trypsinised blood cells was 512) wasapplied to the column (1×30 cm) containing 4 g of macroporouspoly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) with 8% ofbound D-glucose, which was equilibrated with physiologic saline. Aftersoaking of the sample, the column was eluted with saline at the flowrate 8 ml/h and 4 ml fractions were collected. The eluate in fractions3-6 was active in haemaglutination. The elution with 0.2 M solution ofD-glucose in saline was started from the 21st fraction. The fractions23-27 were combined, dialysed for 48 hours against 4×3 l of water, andlyophilised. The yield was 41.1 mg; activity of solution 2048.

EXAMPLE 17

The sample (2 ml) containing 50 mg of Concanavaline A isolated from Jackbeans meal (activity of 1% solution 512) was applied to the column (1×30cm) containing 4 g of macroporous poly(2-hydroxyethylmethacrylate-co-ethylene dimethacrylate) with 8.25 wt.% of boundD-glucose, which was equilibrated with saline. After soaking of thesample, the column was eluted with saline at the flow rate of 8 ml/h and4 ml fractions were collected. No fraction contained the eluate withhaemaglutination activity. The elution with 0.2 M solution of D-glucosein saline was started from the 17th fraction and with the glycine buffersolution of pH 2.7 from the 28th fraction. The fractions 19-22 werecombined, dialysed for 48 hours against 4×3 l of water, and lyophilised.The yield was 46.9 mg; activity of 1% solution against trypsinised bloodcells was slightly higher than 512.

EXAMPLE 18

The copolymer of hydroxyethyl methacrylate with ethylene dimethacrylate(4.2 g, mol. wt. exclusion limit 300,000 daltons) was allowed to swellin 70 ml of 6 wt.% HCl solution in dioxan for 12 hours.N-Acetyl-D-galactosamine (1.046 g) was then added and the suspension wasshaken for 6 hours at laboratory temperature and subsequently for 6hours at 40° C. The reaction mixture was poured into 1 liter ofdeionized water, and washed to neutral reaction and negative reaction onsugars. The copolymer was then washed with ethanol, acetone, and etherand dried in vacuo at 50° C. to the constant weight. The column 1×30 cmcontaining 4 g of the above described derivative with boundN-acetyl-D-galactosamine was equilibrated in physiologic saline. Thesolution of 228.6 mg of the haemaglutination active protein fractionfrom seeds of Dolichos biflorus L. in 3 ml of saline was applied on thecolumn. After soaking of the sample, the column was eluted with salineat the flow rate 10 ml/h and 5 ml fractions were collected. When 155 mlof efluent was collected, the affinity bound protein was eluted with 0.2M N-acetyl-D-galactosamine (5 ml) and then the elution with saline wascontinued. Individual fractions were measured in a UV spectrophotometerat the wave length 280 nm. The fractions containing proteins werecombined (total volume 15 ml), dialysed against 4×2 l of water, andlyophilised. The yield was 17.4 mg; activity of the final productagainst the blood group A₁ was 4095, while that of the original samplewas 128.

EXAMPLE 19

The solution containing 562.7 mg of lyophilised extract from seeds ofRicinus communis in 6 ml of saline was applied to the column (1×30 cm)containing 4 g of macroporous poly(2-hydroxyethyl acrylate-co-ethylenediacrylate) with 14.16% of bound D-galactose, which was equilibratedwith physiologic saline. After soaking of the sample, the column waseluted with saline at the flow rate 8 ml/h and 4 ml fractions werecollected. The eluate in fractions 3-8 was active in haemaglutination.Also the absorbance measured at 280 nm was maximum in these fractions.After elution of the column with 200 ml of saline, the carrier waseluted with 0.2 M D-galactose in saline. The fractions containingprotein were combined (the total was 40 ml), dialysed against 4×4 l ofwater for 48 hours, and the product was lyophilised. The yield was 159.8mg. Activities of 1% solutions against the blood cells of group A₁

    ______________________________________                                        Sulfate fraction before isolation                                                                     4096                                                  Pure protein after isolation                                                                          16,384.                                               ______________________________________                                    

The purity of preparation was checked by an alkaline discontinuouselectrophoresis on polyacrylamide gel.

EXAMPLE 20

The protein fraction (300 mg) from seeds of Ricinus communis wasdissolved in 20 ml of physiologic saline. The solution was titratedagainst the blood cells of group A₁ ; activity in the 12th test-tube was4096. Then, 4 g of the same copolymer with bound D-galactose as inExample 19 was added. The decrease of activity with time was followed.The activity settled after 8 hours. The adsorbent was removed byfiltration and washed with saline. Its whole amount was filled into acolumn, eluted with saline to the disappearance of activity and decreaseof adsorbance to the original value of saline. The bound protein wasthen eluted with 0.2 M solution of D-galactose in physiologic saline.The fractions containing proteins were combined, dialysed against 4×4 lof deionised water for 48 hours, and lyophilised. The yield 238.4 mgcorresponds to the capacity of 59.6 mg per 1 g of the dry copolymer withbound D-galactose.

EXAMPLE 21

The isolation was carried out analogously as in Example 20, with thedistinction that the original amount of protein was 184.4 mg in 20 ml ofphysiologic saline and 3.05 g of the copolymer containing 13.08 wt.% ofD-glucose was used as adsorbent. The yield of pure protein 149.6 mgcorresponds to the capacity of 49.4 mg/g of dry adsorbent.

We claim:
 1. Hydrophilic macroporous three dimensional copolymers ofhydroxyalkyl acrylates or hydroxyalkyl methacrylates, said hydroxyalkylcontaining 1-4 carbon atoms, and containing crosslinking agents inamount of 15 to 50 weight percent selected from the group consisting ofethylene diacrylate and ethylene dimethacrylate, wherein said copolymerscontain, after glycosylation reaction at the surface, saccharides ortheir derivatives, selected from the group consisting ofmonosaccharides, oligosaccharides, deoxy sugars, amino sugars, acylatedsaccharides, ether or halogen derivatives of monosaccharides andoligosaccharides.
 2. A method for producing the copolymers according toclaim 1, wherein the three dimensional copolymers containinghydroxyalkyl groups are reacted with saccharides or their derivatives inan inert organic solvent at a temperature of 20°-100° C. under catalysisof an agent selected from the group consisting of HCl, BF₃, and theirmixtures.